N-Terminal Domains of the Human Telomerase Catalytic Subunit Required for Enzyme Activity in Vivo

Abstract

Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits. However, catalytic activity alone is insufficient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerasenegative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation. We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution. Additionally, we discovered a novel domain, DAT, that "dissociates activities of telomerase," where mutations left the enzyme catalytically active, but was unable to function in vivo. Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.

N-terminal Domain of Yeast Telomerase Reverse Transcriptase: Recruitment of Est3p to the Telomerase Complex

“…The regions showing the highest levels of amino acid conservation among all TERT proteins are coincident with essential regions I, II, and III of yeast TERT (Est2p) identified by Unigenic Evolution ( Figure 1A). These three regions are also essential for human telomerase activity as demonstrated by mutational analysis (Armbruster et al, 2001), supporting the high level of functional conservation between the TERT proteins.…”

“…These phenotypes are consistent with the hypothesis that a substantial portion of region I contributes to recruitment of telomerase accessory proteins. Intriguingly, recent results show that a portion of region I in human TERT (the DAT domain) also contributes to the in vivo activity of telomerase, while being dispensable for catalytic activity (Armbruster et al, 2001). This portion of region I corresponds to residues 51-112 of the S. cerevisiae sequence (using the alignment shown in Supplementary Data).…”

“…This phenotype is reminiscent of that resulting from deletion of EST1 or EST3 and suggests that residues in region I might mediate interactions with telomerase accessory proteins. Intriguingly, mutations within a subdomain of region I of human TERT (the DAT domain) also destroy the in vivo activity of telomerase, but leave catalytic activity intact (Armbruster et al, 2001).…”

Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells

“…This last observation is likely reflective of a gain in the rate of cell cycle progression (see below as well as Figures 7a and b), cumulative over the 2-week period of CFU growth. (DN710), which did not have reverse transcriptase activity, we included the amino-terminal (NDAT92 and NDAT122) and carboxy-terminal (CDAT1127) DAT TERT mutants, which are catalytically active but cannot support telomere maintenance, due to telomere localization defects (Armbruster et al, 2001(Armbruster et al, , 2004Banik et al, 2002), as well as primer-template positioning dysfunctions (Lee et al, 2003). The DAT mutants were previously reported to retain the capacity to promote the non-canonical functions of TERT in chromatin remodeling and are therefore competent to promote DNA-damage repair, despite defects in their canonical TERT functions .…”

“…These defects were rescued by the expression of a recombinant form of TERT, but were not rescued by a catalytically inert TERT mutant . Dissociation-of-activity-in-telomerase (DAT) mutants of TERT, which are catalytically active but cannot support telomere maintenance, also competently restored DNA-damage protection (Armbruster et al, 2001;Banik et al, 2002). Overall, these results suggest that telomerase activity is required for more than just telomere synthesis for cell survival and proliferation.…”